枯草芽胞杆菌谷氨酰胺转氨酶催化蛋白交联过程调控机制的研究

Transglutaminases (TGs) can catalyze the intra- or inter-crosslinking reactions in protein molecules and have been widely applied in food processing to improve the food properties. Currently, only the transglutaminase (MTG) derived from Streptomyces mobaraense is industrially produced and commercialized. However, the single product can hardly fulfill the various processes and substrates of food processing. In the present study, a novel transglutaminase (BTG) from Bacillus subtilis, with superior thermostability and different substrate specificity in comparison with MTG, will be set as the research object. To fully understand the molecular mechanism of substrate specificity, we will analyze the catalytic efficiency of BTG toward different glutamine peptides and figure out the peptides with high catalytic efficiency (Q peptide). Subsequently, three kinds of proteins, respectively with different level of Q pepetide, will be taken as the substrates for cross-linking. The causal and dose-effect relationships on the microstructures, mesoscopic properties, and macroscopic qualities of crosslinks of BTG and proteins in the different processing conditions and parameters will be analyzed to illuminate that the multi-scale mechanism on process of protein-crosslinking catalyzed by BTG. To understand the regulatory mechanism in the BTG-catalyzed processes, the linkage laws of the synergistic effect of some key processing conditions and parameters and multi-scale cross-linking of BTG and proteins will be also investigated. The present research will provide not only fundamental basis on the evaluation of crosslinking effect and optimization of machining process for different proteins, but also new strategies for developing novel TGs and enhancing their abilities of quality control in food processing.

谷氨酰胺转氨酶（TG）可催化蛋白分子内和分子间交联，因此广泛应用于食品加工中改善食品特性。目前只有茂原链轮丝菌TG（MTG）商品化，但其难以适应种类繁多的食品加工底物和过程。本项目以与MTG相比具有不同底物特异性和良好热稳定性的枯草芽胞杆菌TG（BTG）为研究对象，解析BTG对不同谷氨酰胺多肽底物催化效率并筛选高催化效率谷氨酰胺多肽（Q肽），阐明BTG底物特异性分子机制；以含Q肽序列比例高、中、低的三种蛋白分别为底物，解析不同加工条件参数下BTG交联蛋白微观结构、介观性质和宏观品质的因果量效关系，阐明BTG催化蛋白底物交联过程多尺度作用机制；解析关键加工条件参数协同效应与BTG交联蛋白多尺度的联动规律，阐明BTG催化蛋白底物交联过程调控机制。本项目将为BTG针对不同特征蛋白底物交联效果评价、加工过程调控改良提供理论基础和依据，为开发新型TG和增强TG在食品加工过程中品质控制能力提供新策略。

目前只有茂原链轮丝菌TG（MTG）商品化，但其难以适应种类繁多的食品加工底物和过程。枯草芽胞杆菌TG（BTG）与MTG相比具有不同底物特异性和良好热稳定性，可为食品加工业提供新的选择。.本项目（1）对BTG酶学特性进行分析，其最适作用温度为60℃、最适pH为8.0、在60℃以下及pH6.0-9.0活力稳定，表现出良好的热稳定性和pH稳定性；利用mRNA体外展示技术，筛选获得5个与BTG表现出高交联效率的多肽，经分子动力学模拟分析发现酶和多肽底物交联效率的不同主要与结合能相关，从而阐明了BTG底物特异性的分子机制；另外，利用易错PCR结合高通量筛选技术，获得活力及耐热性能提升MTG突变体E164L，经分子动力学模拟分析发现局部区域的不稳定促进了底物分子接近活性位点，从而阐明了MTG高活力的分子机制。（2）利用圆二色谱检测，获得BTG结构与作用温度之间的关系，表明α-螺旋对于维持BTG的酶活性起着十分重要的作用；在不同条件参数下，测定经BTG交联后，牛血清白蛋白、乳清蛋白、大豆分离蛋白形成的粒径、热稳定性、乳化性、发泡性、持水性、质构等情况，获得BTG交联蛋白的微观结构、介观性质和宏观品质之间关联，从而阐明了BTG催化蛋白底物交联过程的作用机制，并确定蛋白底物浓度及温度为影响交联效果的主要因素。（3）对影响交联效果的主要因素-蛋白底物浓度及温度进行调整及控制，通过改善BTG交联蛋白的微观结构、介观性质，进一步提升其宏观品质，从而阐明了BTG催化蛋白底物交联过程的调控机制。.本项目通过对BTG是应用基础研究，阐明了其交联的分子基础、功能特征及调理机制，为其他TG的开发及应用提供了理论指导及技术支撑。